Visualizing mitochondrial heme flow through GAPDH in living cells and its regulation by NO.

Iron protoporphyrin IX (heme) is a redox-active cofactor that is bound in mammalian cells by GAPDH and allocated by a process influenced by physiologic levels of NO. This impacts the activity of many heme proteins including indoleamine dioxygenase-1 (IDO1), a redox enzyme involved in immune response and tumor growth. To gain further understanding we created a tetra-Cys human GAPDH reporter construct (TC-hGAPDH) which after labeling could indicate its heme binding by fluorescence quenching. When purified or expressed in a human cell line, TC-hGAPDH had properties like native GAPDH and heme binding quenched its fluorescence by 45-65%, allowing it to report on GAPDH binding of mitochondrially-generated heme in live cells in real time. In cells with active mitochondrial heme synthesis, low-level NO exposure increased heme allocation to IDO1 while keeping the TC-hGAPDH heme level constant due to replenishment by mitochondria. When mitochondrial heme synthesis was blocked, low NO caused a near complete transfer of the existing heme in TC-hGAPDH to IDO1 in a process that required IDO1 be able to bind the heme and have an active hsp90 present. Higher NO exposure had the opposite effect and caused IDO1 heme to transfer back to TC-hGAPDH. This demonstrated: (i) flow of mitochondrial heme through GAPDH is tightly coupled to target delivery, (ii) NO up- or down-regulates IDO1 activity by promoting a conserved heme exchange with GAPDH that goes in either direction according to the NO exposure level. The ability to drive a concentration-dependent, reversible protein heme exchange is unprecedented and reveals a new role for NO in biology.

Ubiquitination detection techniques.

Ubiquitination is an intricately regulated post-translational modification that involves the covalent attachment of ubiquitin to a substrate protein. The complex dynamic nature of the ubiquitination process regulates diverse cellular functions including targeting proteins for degradation, cell cycle, deoxyribonucleic acid (DNA) damage repair, and numerous cell signaling pathways. Ubiquitination also serves as a crucial mechanism in protein quality control. Dysregulation in ubiquitination could result in lethal disease conditions such as cancers and neurodegenerative diseases. Therefore, the ubiquitination cascade has become an attractive target for therapeutic interventions. Enormous efforts have been made to detect ubiquitination involving different detection techniques to better grasp the underlying molecular mechanisms of ubiquitination. This review discusses a wide range of techniques stretching from the simplest assays to real-time assays. This includes western blotting/immunoblotting, fluorescence assays, chemiluminescence assays, spectrophotometric assays, and nanopore sensing assays. This review compares these applications, and the inherent advantages and limitations.

Fluorescence dequenching assay for the activity of TEV protease.

Tobacco etch virus (TEV) protease is a widely used protease for fusion tag cleavage. Despite its widespread usage, an assay to quickly and easily quantify its activity in laboratory settings is still lacking. Thus, researchers may encounter inefficient cleavage of the desired fusion proteins due to poor activity of a given TEV protease preparation. Here, we describe the development and implementation of a fluorescence dequenching-based assay to quantify TEV protease activity and assess kinetic parameters. The peptide substrate used in this assay consists of a C-terminal TAMRA fluorophore, an N-terminal fluorescein fluorophore, and the canonical TEV protease recognition sequence. The assay is based on a reduction of fluorescence quenching of fluorescein upon cleavage by TEV protease. The substrate peptide was studied spectroscopically to assess feasibility and to propose a plausible mechanism of the assay. The assay was optimized and applied to obtain rapid assessments of TEV protease activity in purified samples and crude lysate extracts. The kinetic data obtained from improved TEV protease variants were compared with a traditional SDS-PAGE assay. Finally, the assay was applied to determine the optimum pH for TEV protease. Further, the study found that the assay is a rapid and simple approach to quantify TEV protease activity. The findings of the assay on crude lysate extracts, activity assay of TEV protease variants, and assessment of optimum pH for TEV protease reactions demonstrate the robust utility of the assay.

Hydrolytically Stable Maleimide-End-Functionalized Polymers for Site-Specific Protein Conjugation.

Site-specific conjugation to cysteines of proteins often uses ester groups to link maleimide or alkene groups to polymers. However, the ester group is susceptible to hydrolysis, potentially losing the benefits gained through bioconjugation. Here, we present a simple conjugation strategy that utilizes the amide bond stability of traditional 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-hydroxysuccinimide coupling while introducing site specificity. Hydrolytically stable maleimide-end-functionalized polymers for site-specific conjugation to free cysteines of proteins were synthesized using reversible addition-fragmentation chain-transfer (RAFT) polymerization. The alpha terminus of the polymers was amidated with a furan-protected aminoethyl maleimide using carbodiimide-based chemistry. Finally, the maleimide was exposed by a retro Diels-Alder reaction to yield the maleimide group, allowing for thiol-maleimide click chemistry for bioconjugation. A thermophilic cellulase from Fervidobacterium nodosum (FnCel5a) was conjugated using various strategies, including random 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)/N-hydroxysuccinimide (NHS) coupling, site-specific hydroxyethyl maleimide (HEMI) end-functionalized coupling, hydroxyethyl acrylate (HEA) end-functionalized coupling, and amidoethyl maleimide (AEMI) end-functionalized coupling. Only the polymers conjugated by EDC and AEMI remained conjugated a week after attachment. This indicates that hydrolytically stable amide-based maleimides are an important bioconjugation strategy for conjugates that require long-term stability, while esters are better suited for systems that require debonding of polymers over time.

2- and N6-functionalized adenosine-5'-diphosphate analogs for the inhibition of mortalin.

Our early efforts to find a covalent inhibitor of mortalin, a member of the 70 kD heat shock protein (Hsp70) family, led us to solve the structure of the mortalin nucleotide-binding domain (NBD) in complex with N6-propargyladenosine-5'-diphosphate. The acquired structure emphasizes the ability of the nucleotide-binding pocket to accommodate modified ADP compounds. A library of ADP analogs modified at either the 2- or N6-positions of adenosine was screened against the mortalin-NBD. Competitive inhibition and binding assays of the analogs demonstrate that modifications at the 2- or N6-positions have potential to bind and inhibit mortalin uniquely compared to other Hsp70 homologs, and that modifications at the 2-position confer the greatest selectivity in binding and inhibition of the mortalin-NBD.

Biophysical Consequences of EVEN-PLUS Syndrome Mutations for the Function of Mortalin.

HSPA9, the gene coding for the mitochondrial chaperone mortalin, is involved in various cellular roles such as mitochondrial protein import, folding, degradation, Fe-S cluster biogenesis, mitochondrial homeostasis, and regulation of the antiapoptotic protein p53. Mutations in the HSPA9 gene, particularly within the region coding for the nucleotide-binding domain (NBD), cause the autosomal disorder known as EVEN-PLUS syndrome. The resulting mutants R126W and Y128C are located on the surface of the mortalin-NBD near the binding interface with the interdomain linker (IDL). We used differential scanning fluorimetry (DSF), biolayer interferometry, X-ray crystallography, ATP hydrolysis assays, and Rosetta docking simulations to study the structural and functional consequences of the EVEN-PLUS syndrome-associated R126W and Y128C mutations within the mortalin-NBD. These results indicate that the surface mutations R126W and Y128C have far-reaching effects that disrupt ATP hydrolysis, interdomain linker binding, and thermostability and increase the propensity for aggregation. The structural differences observed provide insight into how the conformations of mortalin differ from other heat shock protein 70 (Hsp70) homologues. Combined, our biophysical and structural studies contribute to the understanding of the molecular basis for how disease-associated mortalin mutations affect mortalin functionality and the pathogenesis of EVEN-PLUS syndrome.

Biochemical characterization and zinc binding group (ZBGs) inhibition studies on the catalytic domain of MMP7 (cdMMP7).

Matrix metalloproteinase 7 (MMP7/matrilysin-1) has been implicated in many pathological conditions, such as in cancer and inflammatory diseases; therefore, MMP7 has been targeted for drugs. Success in developing a clinical inhibitor, which exhibits suitable specificity and selectivity, will likely require structural and/or kinetic evaluation of enzyme/inhibitor interactions. To enable these future studies we herein describe the over-expression, purification, and characterization of the catalytic domain of MMP7 (cdMMP7). cdMMP7 was over-expressed in an E. coli over-expression system, and the resulting enzyme was processed into inclusion bodies, which were subsequently solubilized, enabling the enzyme to be re-folded into a catalytically-active form. cdMMP7 was shown to bind 1.8eq of Zn(II), exhibit steady-state kinetic constants of 0.4s-1 for kcat and 23µM for Km, and yield CD and fluorescence spectra that are consistent with a properly-folded enzyme. Pre-steady state kinetic studies yielded kinetic mechanisms of cdMMP7, and these mechanisms are similar to those of other MMPs. Inhibition studies on cdMMP7 with four zinc binding group (ZBG) inhibitors showed that maltol, thiomaltol, and allothiomaltol are better inhibitors with lower IC50 values and lower Kd values against cdMMP7 and cdMMP16 than the commonly-used ZBG inhibitor acetohydroxamic acid. Docking studies suggest that improved inhibitory character may be due to interactions with the S1' substrate binding pocket. Finally, a ZnCo-heterobimetallic analog of cdMMP7 with Co(II) bound in the catalytic site was prepared and characterized. This study describes a well-characterized analog of MMP7 that is available for future inhibitor design efforts.

Design, synthesis and evaluation of XZH-5 analogues as STAT3 inhibitors.

Inhibition of the signaling pathways of signal transducer and activator of transcription 3 (STAT 3) has shown to be a promising strategy to combat cancer. In this paper we report the design, synthesis and evaluation of a novel class of small molecule inhibitors, that is, XZH-5 and its analogues, as promising leads for further development of STAT3 inhibitors. Preliminary SARs was established for XZH-5 and its derivatives; and the binding modes were predicted by molecular docking. Lead compounds with IC50 as low as 6.5µM in breast cancer cell lines and 7.6µM in pancreatic cancer cell lines were identified.

Crystal structure of the nucleotide-binding domain of mortalin, the mitochondrial Hsp70 chaperone.

Mortalin, a member of the Hsp70-family of molecular chaperones, functions in a variety of processes including mitochondrial protein import and quality control, Fe-S cluster protein biogenesis, mitochondrial homeostasis, and regulation of p53. Mortalin is implicated in regulation of apoptosis, cell stress response, neurodegeneration, and cancer and is a target of the antitumor compound MKT-077. Like other Hsp70-family members, Mortalin consists of a nucleotide-binding domain (NBD) and a substrate-binding domain. We determined the crystal structure of the NBD of human Mortalin at 2.8 Å resolution. Although the Mortalin nucleotide-binding pocket is highly conserved relative to other Hsp70 family members, we find that its nucleotide affinity is weaker than that of Hsc70. A Parkinson's disease-associated mutation is located on the Mortalin-NBD surface and may contribute to Mortalin aggregation. We present structure-based models for how the Mortalin-NBD may interact with the nucleotide exchange factor GrpEL1, with p53, and with MKT-077. Our structure may contribute to the understanding of disease-associated Mortalin mutations and to improved Mortalin-targeting antitumor compounds.

Molecular basis of antiangiogenic thrombospondin-1 type 1 repeat domain interactions with CD36.

OBJECTIVE: Antiangiogenic activity of thrombospondin-1 and related proteins is mediated by interactions between thrombospondin type 1 repeat (TSR) domains and the CD36, LIMP-2, Emp sequence homology (CLESH) domain of the endothelial cell receptor CD36. We sought to characterize key molecular determinants of the interaction between thrombospondin-1 TSR domains and the CD36 CLESH domain. APPROACH AND RESULTS: Recombinant thrombospondin-1 TSR2 and TSR(2,3) constructs inhibited microvascular endothelial cell migration, microvascular endothelial cell tube formation, and vessel sprouting in aortic ring assays. Interaction with CD36 CLESH decoy peptides negated these effects. Mutational analyses identified a cluster of residues that confer positive charge to the TSR2 surface and mediate interaction with CD36 CLESH. Antiangiogenic activity was significantly reduced by charge-neutralizing mutations of the Arg-Trp ladder in TSR2, but not TSR3. Additionally, I438 and K464 of TSR2 were shown to be required for CD36 CLESH binding to TSR2. A complementary acidic cluster within CD36 CLESH is also required for antiangiogenic activity. CONCLUSIONS: Thrombospondin-1 interacts with CD36 CLESH through electrostatic interactions mediated by a positively charged TSR2 surface and multiple negatively charged CD36 CLESH residues. Two key residues serve as specificity determinants that identify other TSR domains that interact with CD36 CLESH.

RACK1 interacts with filamin-A to regulate plasma membrane levels of the cystic fibrosis transmembrane conductance regulator.

Mutations in cystic fibrosis transmembrane regulator (CFTR), a chloride channel in the apical membranes of secretory epithelial cells, underlie the fatal genetic disorder cystic fibrosis. Certain CFTR mutations, including the common mutation ΔF508-CFTR, result in greatly decreased levels of active CFTR at the apical membrane. Direct interactions between CFTR and the cytoskeletal adaptors filamin-A (FlnA) and Na(+)/H(+) exchanger regulatory factor 1 (NHERF1) stabilize the expression and localization of CFTR at the plasma membrane. The scaffold protein receptor for activated C kinase 1 (RACK1) also stabilizes CFTR surface expression; however, RACK1 does not interact directly with CFTR and its mechanism of action is unknown. In the present study, we report that RACK1 interacts directly with FlnA in vitro and in a Calu-3 airway epithelial cell line. We mapped the interaction between RACK1 and FlnA to the WD4 and WD6 repeats of RACK1 and to a segment of the large rod domain of FlnA, consisting of immunoglobulin-like repeats 8-15. Disruption of the RACK1-FlnA interaction causes a reduction in CFTR surface levels. Our results suggest that a novel RACK1-FlnA interaction is an important regulator of CFTR surface localization.

Structural insights into the conformation and oligomerization of E2~ubiquitin conjugates.

Post-translational modification of proteins by ubiquitin (Ub) regulates a host of cellular processes, including protein quality control, DNA repair, endocytosis, and cellular signaling. In the ubiquitination cascade, a thioester-linked conjugate between the C-terminus of Ub and the active site cysteine of a ubiquitin-conjugating enzyme (E2) is formed. The E2~Ub conjugate interacts with a ubiquitin ligase (E3) to transfer Ub to a lysine residue on a target protein. The flexibly linked E2~Ub conjugates have been shown to form a range of structures in solution. In addition, select E2~Ub conjugates oligomerize through a noncovalent "backside" interaction between Ub and E2 components of different conjugates. Additional studies are needed to bridge the gap between the dynamic monomeric conjugates, E2~Ub oligomers, and the mechanisms of ubiquitination. We present a new 2.35 Å crystal structure of an oligomeric UbcH5c~Ub conjugate. The conjugate forms a staggered linear oligomer that differs substantially from the "infinite spiral" helical arrangement of the only previously reported structure of an oligomeric conjugate. Our structure also differs in intraconjugate conformation from other structurally characterized conjugates. Despite these differences, we find that the backside interaction mode is conserved in different conjugate oligomers and is independent of intraconjugate relative E2-Ub orientations. We delineate a common intraconjugate E2-binding surface on Ub. In addition, we demonstrate that an E3 CHIP (carboxyl terminus of Hsp70 interacting protein) interacts directly with UbcH5c~Ub oligomers, not only with conjugate monomers. These results provide insights into the conformational diversity of E2~Ub conjugates and conjugate oligomers, and into their compatibility and interactions with E3s, which have important consequences for the ubiquitination process.

Structure of filamin A immunoglobulin-like repeat 10 from Homo sapiens.

Filamin A (FlnA) plays a critical role in cytoskeletal organization, cell motility and cellular signaling. FlnA utilizes different binding sites on a series of 24 immunoglobulin-like domains (Ig repeats) to interact with diverse cytosolic proteins and with cytoplasmic portions of membrane proteins. Mutations in a specific domain, Ig10 (FlnA-Ig10), are correlated with two severe forms of the otopalatodigital syndrome spectrum disorders Melnick-Needles syndrome and frontometaphyseal dysplasia. The crystal structure of FlnA-Ig10 determined at 2.44 Šresolution provides insight into the perturbations caused by these mutations.

Expression, purification and structural characterization of functionally replete thrombospondin-1 type 1 repeats in a bacterial expression system.

The matrix glycoprotein thrombospondin-1 (TSP-1) is a prominent regulator of endothelial cells and angiogenesis. The anti-angiogenic and anti-tumorigenic properties of TSP-1 are in part mediated by the TSP-1 type 1 repeat domains 2 and 3, TSR(2,3). Here, we describe the expression and purification of human TSR(2,3) in milligram quantities from an Escherichia coli expression system. Microvascular endothelial cell migration assays and binding assays with a canonical TSP-1 ligand, histidine-rich glycoprotein (HRGP), indicate that recombinant TSR(2,3) exhibits anti-chemotactic and ligand binding properties similar to full length TSP-1. Furthermore, we determined the structure of E. coli expressed TSR(2,3) by X-ray crystallography at 2.4Å and found the structure to be identical to the existing TSR(2,3) crystal structure determined from a Drosophila expression system. The TSR(2,3) expression and purification protocol developed in this study allows for facile expression of TSR(2,3) for biochemical and biophysical studies, and will aid in the elucidation of the molecular mechanisms of TSP-1 anti-angiogenic and anti-tumorigenic activities.

The inducible kinase IKKi is required for IL-17-dependent signaling associated with neutrophilia and pulmonary inflammation.

Interleukin 17 (IL-17) is critical in the pathogenesis of inflammatory and autoimmune diseases. Here we report that Act1, the key adaptor for the IL-17 receptor (IL-7R), formed a complex with the inducible kinase IKKi after stimulation with IL-17. Through the use of IKKi-deficient mice, we found that IKKi was required for IL-17-induced expression of genes encoding inflammatory molecules in primary airway epithelial cells, neutrophilia and pulmonary inflammation. IKKi deficiency abolished IL-17-induced formation of the complex of Act1 and the adaptors TRAF2 and TRAF5, activation of mitogen-activated protein kinases (MAPKs) and mRNA stability, whereas the Act1-TRAF6-transcription factor NF-κB axis was retained. IKKi was required for IL-17-induced phosphorylation of Act1 on Ser311, adjacent to a putative TRAF-binding motif. Substitution of the serine at position 311 with alanine impaired the IL-17-mediated Act1-TRAF2-TRAF5 interaction and gene expression. Thus, IKKi is a kinase newly identified as modulating IL-17 signaling through its effect on Act1 phosphorylation and consequent function.

Biochemical basis of the interaction between cystic fibrosis transmembrane conductance regulator and immunoglobulin-like repeats of filamin.

Mutations in the chloride channel cystic fibrosis transmembrane regulator (CFTR) cause cystic fibrosis, a genetic disorder characterized by defects in CFTR biosynthesis, localization to the cell surface, or activation by regulatory factors. It was discovered recently that surface localization of CFTR is stabilized by an interaction between the CFTR N terminus and the multidomain cytoskeletal protein filamin. The details of the CFTR-filamin interaction, however, are unclear. Using x-ray crystallography, we show how the CFTR N terminus binds to immunoglobulin-like repeat 21 of filamin A (FlnA-Ig21). CFTR binds to beta-strands C and D of FlnA-Ig21 using backbone-backbone hydrogen bonds, a linchpin serine residue, and hydrophobic side-chain packing. We use NMR to determine that the CFTR N terminus also binds to several other immunoglobulin-like repeats from filamin A in vitro. Our structural data explain why the cystic fibrosis-causing S13F mutation disrupts CFTR-filamin interaction. We show that FlnA-Ig repeats transfected into cultured Calu-3 cells disrupt CFTR-filamin interaction and reduce surface levels of CFTR. Our findings suggest that filamin A stabilizes surface CFTR by anchoring it to the actin cytoskeleton through interactions with multiple filamin Ig repeats. Such an interaction mode may allow filamins to cluster multiple CFTR molecules and to promote colocalization of CFTR and other filamin-binding proteins in the apical plasma membrane of epithelial cells.

Structural basis of nucleotide exchange and client binding by the Hsp70 cochaperone Bag2.

Cochaperones are essential for Hsp70- and Hsc70-mediated folding of proteins and include nucleotide-exchange factors (NEFs) that assist protein folding by accelerating ADP-ATP exchange on Hsp70. The cochaperone Bag2 binds misfolded Hsp70 clients and also acts as an NEF, but the molecular basis for its function is unclear. We show that, rather than being a member of the Bag domain family, Bag2 contains a new type of Hsp70 NEF domain, which we call the 'brand new bag' (BNB) domain. Free and Hsc70-bound crystal structures of Bag2-BNB show its dimeric structure, in which a flanking linker helix and loop bind to Hsc70 to promote nucleotide exchange. NMR analysis demonstrates that the client binding sites and Hsc70-interaction sites of the Bag2-BNB overlap, and that Hsc70 can displace clients from Bag2-BNB, indicating a distinct mechanism for the regulation of Hsp70-mediated protein folding by Bag2.

Lipid bilayers: an essential environment for the understanding of membrane proteins.

Membrane protein structure and function is critically dependent on the surrounding environment. Consequently, utilizing a membrane mimetic that adequately models the native membrane environment is essential. A range of membrane mimetics are available but none generates a better model of native aqueous, interfacial, and hydrocarbon core environments than synthetic lipid bilayers. Transmembrane α-helices are very stable in lipid bilayers because of the low water content and low dielectric environment within the bilayer hydrocarbon core that strengthens intrahelical hydrogen bonds and hinders structural rearrangements within the transmembrane helices. Recent evidence from solid-state NMR spectroscopy illustrates that transmembrane α-helices, both in peptides and full-length proteins, appear to be highly uniform based on the observation of resonance patterns in PISEMA spectra. Here, we quantitate for the first time through simulations what we mean by highly uniform structures. Indeed, helices in transmembrane peptides appear to have backbone torsion angles that are uniform within ± 4°. While individual helices can be structurally stable due to intrahelical hydrogen bonds, interhelical interactions within helical bundles can be weak and nonspecific, resulting in multiple packing arrangements. Some helical bundles have the capacity through their amino acid composition for hydrogen bonding and electrostatic interactions to stabilize the interhelical conformations and solid-state NMR data is shown here for both of these situations. Solid-state NMR spectroscopy is unique among the techniques capable of determining three-dimensional structures of proteins in that it provides the ability to characterize structurally the membrane proteins at very high resolution in liquid crystalline lipid bilayers.